Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation

ABSTRACT

Polyhydroxyalkanoate materials are provided which are suitable for repair of soft tissue, augmentation, and as viscosupplements in animals, particularly humans. The materials comprise liquid polyhydroxyalkanoate polymer compositions or polyhydroxyalkanoate microdispersions. Devices also are provided for storage and delivery of the polyhydroxyalkanoate compositions in vivo. Methods are provided for repairing or augmenting soft tissue in animals using the materials. In a preferred embodiment, the method include the steps of (a) selecting the animal soft tissue to be repaired or augmented; and (b) placing an injectable, liquid polyhydroxyalkanoate polymer or a polyhydroxyalkanoate microdispersion into the animal soft tissue, preferably using a minimally-invasive method such as injection. In another embodiment, the liquid polyhydroxyalkanoate polymer compositions or polyhydroxyalkanoate microdispersions are used as viscosupplements.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. provisional application Ser. No. 60/153,810,filed Sep. 14, 1999.

BACKGROUND OF THE INVENTION

The present invention generally relates to injectable liquid forms ormicrodispersions of polymers suitable for use in soft tissue repair,augmentation, and as viscosupplements.

A variety of different materials have been used to repair or augmentsoft tissue defects or to contour abnormalities caused by facialdefects, acne, surgical scarring, trauma or aging. Unfortunately, noneof these materials is considered to be ideal owing to short-comings ineffectiveness or efficacy. For example, liquid silicone was often usedto correct soft tissue defects. However, this material was subsequentlybanned by the FDA when it was discovered that it could migrate todistant parts of the body and cause physiological and clinical problems.Another material, bovine collagen, became available in the 1970's andappeared to be an effective material for treating soft tissue defects.Over time, however, it was discovered that this material was fairlyrapidly absorbed. The rapid resorption was partially solved bycrosslinking the collagen to extend its lifetime to six months; however,frequent injections of the material are still required. Furthermore,allergic reactions due to bovine proteins present in the collagenpersist in the cross-linked material.

A number of newer materials for soft tissue or augmentation have beendescribed. Ceramic particles of calcium phosphate mixed with an aqueousgel carrier in a viscous polymer have been described in U.S. Pat. No.5,204,382 to Wallace et al. However, there appear to be risks associatedwith the use of these nonabsorbable particulate materials relating totheir migration in vivo. Polymers in combination with solvents and athermosetting material with a curing agent have both been proposed byDunn in U.S. Pat. Nos. 4,938,763; 5,278,201; and 5,278,202, but thesolvents necessary to dissolve the polymers appear to be less thanacceptable, and the materials have limited utility in filling softtissue defects because they solidify. Furthermore, these materials andother similar commercial materials have ultimate yield stresses close to10,000 psi compared to between 500 and 2,000 psi for human skin, raisingpulpability concerns and making them too hard for repair of soft tissueand especially for dermal augmentation or repair. Other polymer blendsbased on lactic acid polymers also have been suggested in U.S. Pat. No.4,235,312 to Buchholz.

Other materials for injection, which solidify to serve as bulking agentsor as matrices for tissue ingrowth, are described in U.S. Pat. No.5,709,854 to Vacanti, et al., and PCT/US96/09065 by Reprogenesis.Exemplary materials in the '854 patent include alginate solutions whichare mixed with calcium ions to induce crosslinking after injection. ThePCT application discloses alternative crosslinkable synthetic polymerswhich have similar properties upon exposure to light or multivalentions.

In this case, however, the polymers are considered to be too viscous tobe injected through a needle, which significantly limits their utility.Furthermore, the oligomers also may be slightly soluble in body fluids,facilitating a rapid diffusion out of the site of implantation. Toaddress these concerns, U.S. Pat. Nos. 5,728,752 and 5,824,333 toScopelianos et al., disclose polymers derived from ε-caprolactone,trimethylene carbonate, and/or ether lactones with glycolide, lactideand p-dioxanone units, for use in the repair of soft tissues andaugmentation which have lower viscosities and do not harden afterimplantation. While these compositions appear to have such desirableproperties, these materials are fairly rapidly degraded and thereforewould need to be re-injected at frequent intervals. Moreover, some ofthese polymers break-down to monomers well known to cause undesirableinflammatory responses in vivo.

It is therefore an object of the present invention to provide polymericmaterials for soft tissue repair and augmentation that are safe,injectable, long lasting, bioabsorbable, and biocompatible.

It is a further object of this invention to provide methods forpreparing and using such materials.

SUMMARY OF THE INVENTION

Polyhydroxyalkanoate materials are provided which are suitable forrepair of soft tissue, augmentation, and as viscosupplements in animals,particularly humans. The materials comprise liquid polyhydroxyalkanoatepolymer compositions or polyhydroxyalkanoate microdispersions. Devicesalso are provided for storage and delivery of the polyhydroxyalkanoatecompositions in vivo.

Methods are provided for repairing or augmenting soft tissue in animalsusing the materials. In a preferred embodiment, the method include thesteps of (a) selecting the animal soft tissue to be repaired oraugmented; and (b) placing an injectable, liquid polyhydroxyalkanoatepolymer or a polyhydroxyalkanoate microdispersion into the animal softtissue, preferably using a minimally-invasive method such as injection.In another embodiment, the liquid polyhydroxyalkanoate polymercompositions or polyhydroxyalkanoate microdispersions are used asviscosupplements.

DETAILED DESCRIPTION OF THE INVENTION

It was discovered that polyhydroxyalkanoate polymers can be selectedand/or rendered are suitable for use in soft tissue repair,augmentation, and as viscosupplements. In preferred embodiments, thesepolyhydroxyalkanoate polymer compositions have low viscosities whichenable them to be injected into soft tissue or the knee joint with asyringe and needle. These polymers preferably do not harden afterimplantation. Degradation rates can be controlled so that certaincompositions are slow to bioabsorb, thereby decreasing considerably thefrequency with which the composition must be re-injected.

I. The Polyhydroxyalkanoate Compositions

The composition comprises a fluid material which comprises apolyhydroxyalkanoate. The polyhydroxyalkanoate is either in the form ofa liquid or a microdispersion, and optionally may further include agentsto increase the safety and efficacy of the composition. The PHA must bea fluid at body temperature or must be in the form of a microdispersionsin a fluid carrier.

As used herein, the term “body temperature” refers to the approximateaverage normal, internal temperature of the animal into which thecomposition is to be introduced, for example, about 37° C. in humans.

Physical properties of the compositions which make them useful for theaugmentation of soft tissue are that they can be easily delivered,preferably by injection, to the desired tissue and that the compositionis biocompatible and slowly bioabsorbed.

As used herein, the term “biocompatible” refers to compositions that arewell tolerated by the body and which do not cause a prolonged adverseinflammatory reaction that would affect their function or performance.

As used herein, the term “bioabsorbable” refers to compositions whichdecomposes under normal in vivo physiological conditions into componentswhich can be metabolized or excreted. “Slow bioabsorption” means thatthe composition performs the intended repair, augmentation, orviscosupplementation function for the appropriate time period,preferably longer than 1 month. In contrast, a material that is tooquickly bioabsorbed requires frequent re-injection.

As used herein, the term “microdispersion” refers to a suspension ofparticles. The particles form a separate phase from that of thecontinuous phase. The particles may be in an amorphous or crystallinestate. The particle size and concentration is chosen to provide theappropriate properties of the mixture. Typically, the particle size ison the order of 1 nm to 500 μm.

The compositions preferably can be easily injected using conventionaltechniques, that is, they can be injected manually, such as with asyringe and needle, preferably one having a 16 gauge diameter, morepreferably having a 22 or larger gauge (i.e. smaller diameter needle).

In one embodiment, the PHA is a wax at room temperature (e.g., between20 and 25° C.) which can be heated to body temperature or greater sothat the composition liquefies, rendering it injectable. In a preferredembodiment, the PHA polymers are liquid polymers of polyhydroxyalkanoatecopolymers which do not crystallize at body temperature, which bioabsorbslowly in vivo. Preferably, the material maintains at least half of itsmass or molecular mass for a period over one year after implantation invivo.

Sources of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are a class of naturally occurringpolyesters that are synthesized by numerous organisms in response toenvironmental stress. For reviews, see Byrom, “MiscellaneousBiomaterials,” in Byrom, ed., Biomaterials MacMillan Publishers, London,1991, pp. 333–59; Hocking & Marchessault, “Biopolyesters” in Griffin,ed., Chemistry and Technology of Biodegradable Polymers, Chapman andHall, London, 1994, pp. 48–96; Holmes, “Biologically Produced(R)-3-hydroxyalkanoate Polymers and Copolymers” in Bassett, ed.,Developments in Crystalline Polymers, Elsevier, London, vol. 2, 1988,pp. 1–65; Lafferty et al., “Microbial Production ofPoly-β-hydroxybutyric acid” in Rehm & Reed, eds., Biotechnology,Verlagsgesellschaft, Weinheim, vol. 66, 1988, pp. 135–76; Müller &Seebach, Angew. Chem. Int. Ed. Engl. 32:477–502 (1993); Steinbüchel,“Polyhydroxyalkanoic Acids” in Byrom, ed., Biomaterials, MacMillanPublishers, London, 1991, pp. 123–213; Williams & Peoples, CHEMTECH,26:38–44, (1996), and the recent review by Madison & Husiman, Microbiol.& Mol. Biol. Rev. 63:21–53 (1999).

The PHA biopolymers may be broadly divided into three groups accordingto the length of their pendant groups and their respective biosyntheticpathways. Those with short pendant groups, such as polyhydroxybutyrate(PHB), a homopolymer of R-3-hydroxybutyric acid (R-3HB) units, arehighly crystalline thermoplastic materials, and have been known thelongest (Lemoigne & Roukhelman, Annales des fermentations, 5:527–36(1925)). A second group of PHAs containing the short R-3HB unitsrandomly polymerized with much longer pendant group hydroxy acid unitswere first reported in the early seventies (Wallen & Rohwedder, Environ.Sci. Technol, 8:576–79 (1974)). A number of microorganisms whichspecifically produce copolymers of R-3HB with these longer pendant grouphydroxy acid units are also known and belong to this second group(Steinbüchel & Wiese, Appl. Microbiol. Biotechnol., 37:691–97 (1992)).In the early eighties, a research group in The Netherlands identified athird group of PHAs, which contained predominantly longer pendant grouphydroxy acids (De Smet, et al., J. Bacteriol., 154:870–78 (1983)).

The PHA polymers may constitute up to 90% of the dry cell weight ofbacteria, and are found as discrete granules inside the bacterial cells.These PHA granules accumulate in response to nutrient limitation andserve as carbon and energy reserve materials. Distinct pathways are usedby microorganisms to produce each group of these polymers. One of thesepathways leading to the short pendant group polyhydroxyalkanoates(SPGPHAs) involves three enzymes, namely thiolase, reductase and PHBsynthase (sometimes called polymerase). Using this pathway, thehomopolymer PHB is synthesized by condensation of two molecules ofacetyl-Coenzyme A to give acetoacetyl-Coenzyme A, followed by reductionof this intermediate to R-3-hydroxybutyryl-Coenzyme A, and subsequentpolymerization. The last enzyme in this pathway, namely the synthase,has a substrate specificity that can accommodate C3–C5 monomeric unitsincluding R-4-hydroxy acid and R-5-hydroxy acid units. This biosyntheticpathway is found, for example, in the bacteria Zoogloea ramigera andAlcaligenes eutrophus.

The biosynthetic pathway which is used to make the third group of PHAs,namely the long pendant group polyhydroxyalkanoates (LPGPHAs), is stillpartly unknown, however, it is currently thought that the monomerichydroxyacyl units leading to the LPGPHAs are derived by the β-oxidationof fatty acids and the fatty acid pathway. The R-3-hydroxyacyl-Coenzymesubstrates resulting from these routes are then polymerized by PHAsynthases (sometimes called polymerases) that have substratespecificities favoring the larger monomeric units in the C6–C14 range.Long pendant group PHAs are produced, for example, by Pseudomonads.

Presumably, the second group of PHAs containing both short R-3HB unitsand longer pendant group monomers utilize both the pathways describedabove to provide the hydroxy acid monomers. The latter are thenpolymerized by PHA synthases able to accept these units.

In all about 100 different types of hydroxy acids have been incorporatedinto PHAs by fermentation methods so far (Williams, et. al., Int. J.Biol. Macromol., 25:111–21 (1999)). Notably, these include PHAscontaining functionalized pendant groups such as esters, double bonds,alkoxy, aromatic, halogens and hydroxy groups.

During the mid-1980's, several research groups were actively identifyingand isolating the genes and gene products responsible for PHA synthesis.These efforts have lead to the development of transgenic systems forproduction of PHAs in both microorganism and plants, as well asenzymatic methods for PHA synthesis. Such routes could increase furtherthe available PHA types. These advances have been reviewed in Williams &Peoples, CHEMTECH, 26:38–44 (1996), Madison & Huisman, Microbiol. Mol.Biol. Rev., 63:21–53 (1999), and Williams & Peoples, Chem. Br. 33:29–32(1997).

In addition to using biological routes for PHA synthesis, PHA polymersmay also be derived by chemical synthesis. One widely used approachinvolves the ring-opening polymerization of β-lactone monomers usingvarious catalysts or initiators such as aluminoxanes, distannoxanes, oralkoxy-zinc and alkoxy-aluminum compounds (see Agostini, et al., Polym.Sci., Part A-1, 9:2775–87 (1971); Gross, et al., Macromolecules,21:2657–68 (1988); Dubois, et al., Macromolecules, 26:4407–12 (1993); LeBorgne & Spassky, Polymer, 30:2312–19 (1989); Tanahashi & Doi,Macromolecules, 24:5732–33 (1991); Hori, et al., Macromolecules,26:4388–90 (1993); Kemnitzer, et al., Macromolecules, 26:1221–29 (1993);Hori, et al., Macromolecules, 26:5533–34 (1993); Hocking & Marchessault,Polym. Bull., 30:163–70 (1993); U.S. Pat. Nos. 5,489,470 and 5,502,116to Noda). A second approach involves condensation polymerization ofesters and is described in U.S. Pat. No. 5,563,239 to Hubbs, et al., andreferences therein. Researchers also have developed chemo-enzymaticmethods to prepare PHAs. Xie et al., Macromolecules, 30:6997–98 (1997),for example, discloses a ring opening polymerization ofbeta-butyrolactone by thermophilic lipases to yield PHB.

The polyhydroxyalkanoates are also generally available in two physicalforms, namely a latex form (Koosha, F. Ph.D. Dissertation, 1989, Univ.Nottingham, UK., Diss. Abstr. Int. B 51:1206 (1990)), and as a drypowder. Polyhydroxyalkanoates useful in the present compositions can bederived using any of the above methods, alone or in conjunction with thetechniques described in the Examples below.

(i) Liquid Polymers

The polyhydroxyalkanoate liquid copolymers may contain varying amountsof the different hydroxy acid monomer types depending upon the specificproperties that the liquid copolymer is desired to have. Thesepolyhydroxyalkanoate polymers also may be blended with otherpolyhydroxyalkanoate polymers or other suitable materials prior to use.

In a preferred embodiment, the polymer is derived from one or moremonomers selected from the group consisting of 2-hydroxybutanoate,3-hydroxyalkanoates, 3-hydroxyalkenoates, 4-hydroxyalkanoates,4-hydroxyalkenoates, 5-hydroxyalkanoates, 5-hydroxyalkenoates,6-hydroxyalkanoates, and 6-hydroxyalkenoates. Preferred species includehomopolymers and copolymers containing any combination of the monomers3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxypropionate,2-hydroxybutyrate, 4-hydroxybutyrate, 4-hydroxyvalerate,3-hydroxyhexanaote, 3-hydroxyheptanoate, 3-hydroxyoctanaote,3-hydroxynonanoate, 3-hydroxydecanoate, 3-hydroxyundecanoate,3-hydroxydodecanoate, 3-hydroxytridecanoate, 3-hydroxytetradecanoate,3-hydroxypentadecanoate, 3-hydroxyhexadecanoate, 3-hydroxyheptadecanoateand 3-hydroxyoctadecanoate.

The viscosity of the liquid polyhydroxyalkanoate polymers may be variedby changing the molecular weight of the polymer, crosslinking, and/or bychanging the composition of the polymers. The desired molecular weightmay be achieved during the initial polymer synthesis, or alternativelyadjusted up or down subsequently. Suitable methods for decreasing themolecular weight of polyhydroxyalkanoates, particularly to convert themfrom solid to liquid forms, include hydrolysis (particularly using acidcatalysis), enzymatic degradation, irradiation, and mechanical orthermal treatments. Particularly desirable PHAs have relatively lowmolecular weights and are amorphous. Preferred PHA molecular weights aregenerally less than 100,000, preferably less than 50,000.Polyhydroxyalkanoates may be crosslinked by methods including the use ofradical chemistry, irradiation, and with crosslinking agents.Representative methods are described in de Koning et al., Polymer,35:2090–97 (1994); Gagnon, et al., Polymer, 35:4358–67 (1994); andGagnon, et al., Polymer, 35:4368–75 (1994). Certainpolyhydroxyalkanoates may also contain functionalities in their pendantgroups such as unsaturation which can be a preferred site ofcrosslinking. Preferred polyhydroxyalkanoate polymer compositions willhave viscosities high enough to prevent them from being dissolved inbodily fluids, but low enough to allow them to be easily injected.

A suitable viscosity would allow manual injection of the fluid through a16 g needle, and a preferred viscosity would allow manual injection ofthe fluid through a 22 or smaller gauge needle. A suitable range ofviscosity would be less than about 1,000,000 cP. The preferred range ofviscosity varies from that of water (1 cP) to about that of molasses(100,000 cP). The viscosity of a fluid typically depends on thetemperature, thus it is possible to adjust the viscosity of a fluid byvarying its temperature. Typically viscosity of a material is lower athigher temperature. Prior to injection, the temperature of the fluid maybe increased to facilitate injection. Depending upon the particleconcentration, colloidal suspensions of PHA particles can be producedwhich have low viscosity (<100 000 cP). Absorption of the carrier fluidfrom the tissue can result in aggregation and potential coalescence ofPHA particles. Depending on the composition and Mw, PHA polymers in theliquid form can be produced with higher viscosities (>100 000 cP). Priorto injection, the temperature of the liquid PHA may be increased tofacilitate injection.

In addition to controlling viscosity by altering composition, molecularweight and using crosslinking, it is also possible to use these methodsto control the rate of bioabsorption of the injectablepolyhydroxyalkanoate in vivo. It is therefore possible to tailorbioabsorption rates to an application.

For medical or veterinary use, the PHA polymers may be sterilized, forexample by gamma irradiation or by using ethylene oxide. Certain PHApolymers also may be sterilized in an autoclave with steam.

(ii) Polymer Microdispersions

In this embodiment of the compositions, suitable fluid carriers includeliquid polyhydroxyalkanoates and aqueous solutions. Suitablepolyhydroxyalkanoate particulates will have a diameter less than about500 μm, preferably less than 50 μm, and most preferably less than about5 μm.

The dispersed particles may be in a semi-crystalline or a fluid-likeamorphous state. The amorphous state is preferred when aggregation andcoalesce of the particle into larger particles is desired. Aggregationand coalescence is likely to be facilitated by absorption of the fluidcarrier. Larger aggregates are often preferred as they are less likelyto migrate from the site of injection. Larger or smaller particles maybe preferred when surface area affects the bioabsorption profile.

Semicrystalline particles are preferred when the properties of thecrystalline phase are desired. Semicrystalline particles are expected tohave a longer absorption profile than analogous amorphous particles.Additionally, the diffusion of added agents (such as drugs or bioactivecompounds) is affected by the crystalline state of the material.Diffusion of a drug out of a semicrystalline particle is typicallyslower than from an analogous amorphous particle. Thus, crystallinitymay be adjusted to optimize the release of an added drug. Amicrodispersion may be preferred over a liquid polymer when lowviscosity is desired. PHA microdispersions containing high solidsconcentration (>10% by weight solids) can be prepared which are suitablefor injection. A liquid polymer would be preferred when the use of acarrier fluid is undesirable or when the polymer is intended to form adense deposit.

Other Agents

The compositions may further include other agents to increase the safetyand efficacy of the composition. Examples of such agents includecompounds with anti-microbial activity anesthetics, adjuvants,anti-inflammatory compounds, surfactants, steroids, lipids, enzymes,antibodies, and hormones.

Other agents which can be included in the compositions includepharmacologically active or bioactive compounds, and dyes. Proteins andpeptides can be included.

II. Applications for the Polyhydroxyalkanoate Compositions

The polyhydroxyalkanoate compositions may be administered anywhere inthe body of animals where a bulking agent or viscosupplement is needed(e.g., intradermally, subcutaneously, intramuscularly and submucosally)in a therapeutic amount to provide the desired cosmetic, prosthetic, orpain-relieving effect. As used herein, the term “animal” includesmammals, preferably humans.

The compositions can be used for a variety of soft tissue repair andaugmentation procedures, and as viscosupplements. For example, they canbe used in facial tissue repair or augmentation including, but notlimited to, camouflaging scars, filling depressions, smoothing outirregularities, correcting asymmetry in facial hemiatrophy, secondbranchial arch syndrome, facial lipodystrophy and camouflagingage-related wrinkles as well as augmenting facial eminences (lips,brows, etc.). Additionally, these compositions can be used to restore orimprove sphincter function such as for treating stress urinaryincontinence. Other uses include the treatment of vesicoureteral refluxby subureteric injection and application of these materials as generalpurpose fillers in the human body.

The compositions can be used a surgical aid.

In one embodiment, the compositions may be injected into skeletaltissues, such as bone, cartilage, tendons, and muscles. Such embodimentscan be used to facilitate tissue repair or regeneration.

The compositions may also be used as viscosupplements, for example, torelieve pain due to osteoarthritis of the knee, in a similar manner tothe commercial use of the product SYNVISC™. By directly injecting one ofthe presently described polyhydroxyalkanoate compositions into the kneejoint, the material can act as a shock absorber and lubricant, providingprolonged relief from pain.

Representative surgical applications for the compositions include facialcontouring (frown or glabellar line, acne scars, cheek depressions,vertical or perioral lip lines, marionette lines or oral commissures,worry or forehead lines, crow's feet or peri-orbital lines, deep smilelines or nasolabial folds, smile lines, facial scars, lips and thelike); periurethral injection including injection into the submucosa ofthe urethra along the urethra, at or around the urethral-bladderjunction to the external sphincter; ureteral injection for theprevention of urinary reflux; injection into the tissues of thegastrointestinal tract for the bulking of tissue to prevent reflux; toaid in sphincter muscle coaptation, internal or external, and forcoaptation of an enlarged lumen; intraocular injection for thereplacement of vitreous fluid or maintenance of intraocular pressure forretinal detachment; injection into anatomical ducts to temporarily plugthe outlet to prevent reflux or infection propagation; larynxrehabilitation after surgery or atrophy; and any other soft tissue thatcan be augmented for cosmetic or therapeutic affect.

III. Methods and Devices for Administration of the Compositions

A variety of devices may be used for administering the compositions. Apreferred method of administration uses a syringe and needle. Othersuitable devices include the carpule device described in U.S. Pat. Nos.4,664,655 and 4,758,234. Additional means may also be used to facilitatedelivery of highly viscous polyhydroxyalkanoate compositions, such asthe use of powered devices and devices which heat the polymercomposition prior to delivery.

In one embodiment, the polyhydroxyalkanoate compositions are provided inthe form of a kit including the polymeric materials in a reservoir alongwith delivery means, for example, a syringe or catheter.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Injectable Compositions of Polyhydroxyalkanoate 3836

PHA3836 (poly-3-hydroxyoctanoate-co-3-hydroxyhexanoate) (6.75 g, Mw100,000), derived by microbial fermentation, was dissolved in dioxane(90 ml) containing 10 ml of concentrated hydrochloric acid.1,3-Butanediol (2.5 ml) was added and the mixture was heated to reflux.Samples (5 ml) were removed periodically and dried by rotaryevaporation. The molecular mass of these products were determined by GPCanalysis, see Table 1. Lower molecular weight compositions of PHA3836were found to be suitable for injection as viscous fluids, particularlyafter heating prior to injection.

TABLE 1 Molecular Weight Analysis of Acid Alkoholysis Products ofPHA3836 Reaction Time (min.) Mw^(a) Mw/Mn^(a) 0 103000 2.7 20 29000 1.840 7400 1.6 60 4700 1.7 80 2900 1.5 100 2600 1.5 120 2000 1.5 180 15001.5 240 1200 1.4 ^(a)Determined by GPC analysis. Isolated polymers weredissolved in chloroform at approximately 1 mg/mL and samples (50 μL)were chromatographed on a Waters Stryagel HT6E column at a flow rate of1 mL chloroform per minute at room temperature using a refractive indexdetector. Molecular masses were determined relative to polystyrenestandards of narrow polydispersity.

EXAMPLE 2 Injectable Polyhydroxyalkanoate 3836 Compositions

PHA3836 (20.0 g, Mw 100,000) was dissolved in dioxane (250 ml) withheating. After complete dissolution, concentrated hydrochloric acid (20ml) and 1,3-butanediol (10 g) were added. The mixture was heated toreflux. Samples (100 ml) were removed at 10 minutes and 30 minutes,samples A and B, respectively. Solvent was removed by rotary evaporationfollowed by lyophilization. The molecular mass of these products weredetermined by GPC analysis, see Table 2. Upon standing at roomtemperature, samples A and B solidified into elastic, waxy materials.After heating to 50° C., these waxy materials became viscous fluidssuitable for injection. After cooling to room temperature, products Aand B remained viscous fluids.

TABLE 2 Molecular Weight Analysis of Acid Alcoholysis Products ofPHA3836 Sample Reaction Time (min.) Mw^(a) Mw/Mn^(a) A 10 60000 2.0 B 3020000 1.8 ^(a)Determined by GPC analysis, see Table 1 for GPCconditions.

EXAMPLE 3 Injectable Polyhydroxyalkanoate 3836 Compositions

PHA3836 (30.0 g, Mw 100,000) was dissolved in dioxane (200 ml) withheating. After complete dissolution, concentrated hydrochloric acid (20ml) was added and the mixture was heated to reflux for 40 minutes. Aftercooling to room temperature, solid sodium bicarbonate was added toneutralize the acid. Solid MgSO₄ was added to remove water. The mixturewas filtered to remove solids and concentrated to yield the oligomericPHA3836 (27 g, yield 90%). This material was designated as sample C. Themolecular mass of this product was determined to be 15,000 by GPCanalysis, see Table 1 for GPC conditions. Upon standing at roomtemperature, sample C became an elastic, waxy material. After heating to50° C., the wax became a viscous fluid suitable for injection.

EXAMPLE 4 Injectable Polyhydroxyalkanoate 3400 Compositions

PHA3400 (poly-3-hydroxybutyrate) was dissolved in glacial acetic acid byheating at reflux with overhead stirring to yield a 6% solution. Aftercomplete dissolution of PHA3400, water (15% of the acetic acid volume)was added to yield a clear solution. Initially, the solution is viscous,however, with time the viscosity decreases as the Mw of the polymer isreduced. The solution was stirred at reflux (108° C.). At various times,aliquots (3 ml) were removed and were precipitated into water (10 ml).The precipitate was collected via filtration, washed with water, driedand analyzed. The precipitate was weighed to determine the amount ofprecipitated material. The yields of recoverable material weretypically >60%, thus a significant portion of PHA3400 may be soluble inthe acetic acid/water solution after precipitation of the polymer. TheMw of the polymer was analyzed by GPC, using a column which is designedfor the analysis of low Mw polymers (500 000–500 g/mol, Waters HR4E).The analysis was performed in chloroform (1 ml/min, RI detector, ambienttemp., polystyrene standards), see Table 3.

The hydrolysis of PHA3400 in 85% acetic acid at reflux (108° C.)proceeds smoothly with reasonable recovery of product (>60%). A plot oflog Mw versus log Reaction Time is linear, thus one can optimize theprocess to yield polymer of desired Mw and viscosity by varying thereaction time. There is no crotonization of the polymer and theresultant product is partially terminally acetylated, as evidenced bythe acetyl resonance in the ¹H NMR spectrum. Under the conditions used,polymer of 8,000 g/mol can be produced in 4 hours and 1,500 g/mol(Mw/Mn=1.32) can be produced in about 23 hours.

In general the PHA3400 oligomers are semi-crystalline at roomtemperature. These materials can be melt or solution mixed with avariety of other biocompatible materials or solvents to yield viscousfluids at body temperature that are suitable for injection into softtissue.

TABLE 3 Data for PHA3400 hydrolysis in 85% Acetic Acid Reflux time (hr)Mass (mg) Yield % Mw  0 137 80 354,000  1 69 40  2 102 59 16,000  4 10863 8,000  8 118 69 4,400 23 (end) 9.5 g 60 1,500

EXAMPLE 5 Injectable Polyhydroxyalkanoate 4400 Compositions

Dissolve PHA4400 (poly-4-hydroxybutyrate, 8.5 g, Mw 430,000) inanhydrous THF (280 ml) to produce 3% wt./vol. solution. Apply gentleheating to 60° C. to facilitate dissolution of the polymer. Slowly add 1ml of absolute ethanol. Cool solution to room temperature. Add aliquotsof sodium methoxide (0.1 M in methanol) to provide desired Mw andviscosity of the product (see Table 4). Stir at room temperature for 10minutes. Quench reaction with acid (if desired). Filter and evaporateTHF to yield product (7.5 g, for 300 μL added sodium methoxide).

In general the PHA4400 oligomers are semi-crystalline at roomtemperature. These materials can be melt or solution mixed with avariety of other biocompatible materials or solvents to yield viscousfluids at body temperature that are suitable for injection into softtissue.

TABLE 4 Data for PHA4400 Hydrolysis Amount added MeONa (μL) GPC Ret.Time (min.) Molecular Mass^(b) O (starting material) 7.87 430,000 1008.0 320,000 200 8.6 82,000 300 9.1 25,000 ^(b)Log Mw = GPC Ret. Time *(−0.984) + 13.376, determined relative to polystyrene.

EXAMPLE 6 Injectable Polyhydroxyalkanoate 3444 Compositions

PHA3444 (poly-3-hydroxybutyrate-co-4-hydroxybutyrate) copolymers wereprepared in recombinant E. coli. The polymers were extracted from thedried biomass with chloroform and precipitated into 3–5 volumes ofmethanol. The material properties of these copolymers can be varieddepending on the monomeric composition of polymers, see Table 5.Copolymers containing greater than 10% 4-hydroxybutyrate (4HB) wereelastic and rubbery. Samples containing 30–35% 4-hydroxybutyrate were oflow crystallinity as demonstrated by low DH, and were slow torecrystallize from the melt.

In general the PHA3444 oligomers are semi-crystalline at roomtemperature. The amount of crystallinity can be adjusted by varying thecomposition. These materials can be melt or solution mixed with avariety of other biocompatible materials or solvents to yield viscousfluids at body temperature that are suitable for injection into softtissue.

TABLE 5 Properties of PHA3444 Copolymers Produced in Recombinant E. coli% 4HB DH (J/g) Tg (° C.) Mw (by GPC) 12 60 −7 760,000 15 44 −10 830,00032 10 −20 800,000

Modifications and variations of the compositions and methods of use willbe obvious to those skilled in the art from the foregoing detaileddescription and are intended to come with the following claims.

1. A composition for the repair or augmentation of tissue in animal orhuman, comprising: a biocompatible, bioabsorbable fluid which comprisesa polyhydroxyalkanoate which is injectable into a human or animal forrepair or augmentation of tissue, wherein the polyhydroxyalkanoate is aliquid or wax at a temperature between about 20 and 25° C. or whereinthe polyhydroxyalkanoate is liquid at the body temperature of theanimal.
 2. The composition of claim 1 wherein the polyhydroxyalkanoateis a liquid at about 37° C.
 3. The composition of claim 1 wherein thebiocompatible fluid is a microdispersion of particles of thepolyhydroxyalkanoate dispersed in a physiologically compatible liquidcarrier.
 4. The composition of claim 3 wherein the carrier is a secondpolyhydroxyalkanoate or an aqueous solution.
 5. The composition of claim1 wherein the particles have a diameter of less than about 500 μm. 6.The composition of claim 5 wherein the diameter is less than about 50μm.
 7. The composition of claim 6 wherein the diameter is less thanabout 5 μm.
 8. The composition of claim 1 wherein the polymer derivedfrom one or more monomers selected from the group consisting of2-hydroxybutanoate, 3-hydroxyalkanoates, 3-hydroxyalkenoates,4-hydroxyalkanoates, 4-hydroxyalkenoates, 5-hydroxyalkanoates,5-hydroxyalkenoates, 6-hydroxyalkanoates, and 6-hydroxyalkenoates. 9.The composition of claim 1 wherein the polyhydroxyalkanoate has amolecular weight of less than 100,000 as determined by gel permeationchromatography.
 10. The composition of claim 9 wherein the molecularweight is less than 50,000 as determined by gas permeationchromatography.
 11. The composition of claim 1 having a viscosity ofbetween about 1 and 100,000 cP.
 12. The composition of claim 11 having aviscosity of between about 1 and 10,000 cP.
 13. The composition of claim1 further comprising a bioactive agent.
 14. The composition of claim 1further comprising a peptide or protein.
 15. The composition of claim 1wherein the polyhydroxyalkanoate is amorphous.
 16. A compositionsuitable for use in the treatment of osteoarthritic knees comprising: abiocompatible, bioabsorbable fluid which comprises apolyhydroxyalkanoate, wherein the composition is suitable for use as aviscosupplement, wherein the polyhydroxyalkanoate is a liquid or wax ata temperature between about 20 and 25° C. or wherein thepolyhydroxyalkanoate is liquid at the body temperature of the animal.17. A kit comprising (a) the composition of claim 1; and (b) a means fordelivering the composition to a patient.
 18. The kit of claim 17 whereinthe means for delivering comprises a needle and a syringe.